U.S. patent number 5,340,747 [Application Number 08/072,346] was granted by the patent office on 1994-08-23 for diagnostic microbiological testing apparatus and method.
This patent grant is currently assigned to Difco Laboratories, Inc.. Invention is credited to Gideon Eden.
United States Patent |
5,340,747 |
Eden |
August 23, 1994 |
Diagnostic microbiological testing apparatus and method
Abstract
A diagnostic microbiological testing apparatus and method
includes at least one test tray including a plurality of reaction
chambers, a light source disposed proximate to the test tray for
directing light, at an excitation wavelength of a fluorescence
emitting agent contained within the reaction chambers, at the test
tray, a filter for passing therethrough only light generated by a
fluorescence emitting reaction resulting from the interaction of
the fluorescence emitting agent and a sample, and an imaging
mechanism for detecting only the light generated by the
fluorescence emitting reaction at the emission wavelength
simultaneously from the plurality of reaction chambers.
Inventors: |
Eden; Gideon (Ann Arbor,
MI) |
Assignee: |
Difco Laboratories, Inc. (Ann
Arbor, MI)
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Family
ID: |
25302305 |
Appl.
No.: |
08/072,346 |
Filed: |
June 4, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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848087 |
Mar 9, 1992 |
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Current U.S.
Class: |
436/172;
250/361C; 250/461.2; 422/52; 422/561; 422/64; 422/82.08; 422/82.09;
435/287.3; 435/288.7; 435/808; 435/809; 436/45; 436/46;
436/807 |
Current CPC
Class: |
G01N
21/253 (20130101); C12M 41/46 (20130101); G01N
21/6428 (20130101); G01N 2035/00118 (20130101); G01N
2035/00148 (20130101); G01N 2201/0415 (20130101); Y10S
435/808 (20130101); Y10S 436/807 (20130101); Y10S
435/809 (20130101); Y10T 436/112499 (20150115); Y10T
436/111666 (20150115) |
Current International
Class: |
C12M
1/34 (20060101); G01N 21/25 (20060101); G01N
35/00 (20060101); G01N 33/487 (20060101); C12M
001/34 (); G01N 021/64 (); G01N 021/76 (); G01N
035/02 () |
Field of
Search: |
;435/29,289,291,808,809
;422/52,58,63,64,67,82.08,82.09 ;436/46,45,172,800,807
;250/361C,461.2,328 ;356/417 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Warden; Robert J.
Assistant Examiner: Crawford; L. M.
Attorney, Agent or Firm: Reising, Ethington, Barnard, Perry
& Milton
Parent Case Text
This is a continuation-in-part of Ser. No. 07/848,087 filed on Mar.
9, 1992, now abandoned.
Claims
What is claimed is:
1. A diagnostic microbiological testing apparatus for detecting the
presence of fluorescence emitting reaction (FER) resulting from the
interaction of fluorescence emitting agents (FEA) and a sample for
susceptibility and identification testing, said apparatus
consisting essentially of: a test tray including a plurality of
reaction chambers containing an FEA, which upon reaction with a
predetermined microbe in a sample will emit light at a
predetermined emission wavelength upon being illuminated by light
at a predetermined excitation wavelength;
a narrow band light source disposed proximate to said plurality of
reaction chambers of said test tray for directing light
substantially at said excitation wavelength at said plurality of
reaction chambers;
filter means for passing therethrough only light generated by the
FER at the emission wavelength; and a video means for detecting
only the light generated by an FER at the emission wavelength
simultaneously from all the said plurality of reaction chambers of
said test tray and forming an image thereof, said filter means
being disposed between said test tray and video means.
2. An apparatus as set forth in claim 1 further including a
detection area, carousel means for containing a plurality of said
test trays, indexing means for sequentially moving said carousel to
selectively position each of said test trays proximate to said
detection area, and positioning means for positioning one of said
test trays proximate to said detection area into and out of said
detection area.
3. An apparatus as set forth in claim 1 further including reaction
media for reacting with a predetermined microbe to emit the light
at said emission wavelength, said video means detecting the
presence of the microbe by detecting said light at said emission
wavelength.
4. An apparatus as set forth in claim 1 wherein said reaction
chambers further include an antibiotic for susceptibility
testing.
5. A method of detecting the presence of a fluorescence emitting
reaction (FER) resulting from the interaction of fluorescence
emitting agents (FEA) and a sample for susceptibility and
identification testing, said method consisting essentially of the
steps of: containing an FEA in a plurality of reaction chambers of
a test tray, the FEA upon reaction with a predetermined microbe in
a sample emitting light at a predetermined emission wavelength upon
being illuminated by light at a predetermined excitation
wavelength;
directing a narrow band light substantially at the excitation
wavelength at the plurality of reaction chambers of the test
tray
passing through a filter only light generated by an FER at the
emission wavelength simultaneously from the plurality of reaction
chambers; and video imaging only the passed through light generated
by the FER at the emission wavelength simultaneously from all of
the plurality of reaction chambers of the test tray.
Description
TECHNICAL FIELD
The present invention relates to microbiological testing apparatus
and methods and, more specifically, to means for susceptibility and
identification testing of samples, such as those from patients
possibly infected by a microbe.
BACKGROUND OF THE INVENTION
Many systems exist for conducting tests of microbiological samples
for providing patient diagnosis and therapy. It is desirable to use
automated systems requiring minimal handling by a technician. At
the same time, it is also desirable to utilize systems which
provide the most accurate results possible.
Such systems as described above can be used for identification
testing wherein it is desirable to determine the identification of
any microbes present in a patient's sample. Alternatively, or
additionally, it is also desirable to utilize a systems which can
be used for susceptibility testing. Susceptibility testing
determines the susceptibility of a microbe in a sample to various
therapeutics, such as antibiotics.
The U.S. Pat. No. 3,297,873 to Hovnanian discloses a television
camera utilized with a control unit in video amplifier, a
horizontal line analyzer and a TV monitor. This provides a visual
read-out of micro-organisms. The TV monitor displays the specimen
or a part of the specimen utilizing a lens and filter combination.
Furthermore, a photometer determines the UV absorption or
transmission characteristics of micro-organisms, cells or other
micro-specimens. The display includes a darkened area, as well as a
display of area. The darkened area represents the micro-sampled
portion of the specimen.
The U.S. Pat. No. 4,061,469 to DuBose discloses a blood analyzer
which utilizes two photodetectors, one as the measuring detector of
the sample and the other as a reference detector. The second
photodetector senses energy supplied from the source through an
individual sample.
The U.S. Pat. No. 4,166,095 to Kling discloses an automatic
chemical testing apparatus with visual monitoring and inputting of
test results.
The U.S. Pat. No. 4,175,860 to Bacus discloses a method and
apparatus for classifying cells, such as red blood cells. The
apparatus generates an image that is split into a high resolution
and a low resolution image wherein the circuitry performs
measurement and analysis relating to the size, density and color of
the cytoplasm and the nucleus. The analysis obtained from each of
the two images are applied to classification logic circuitry for
the purpose of determining malignant cells. The images are obtained
from a ridicom camera which are sent to an analog digital converter
and to a video monitor. A single slide is used.
The U.S. Pat. No. 4,431,307 to Suovaniemi discloses a particular
type of cuvette whose slide walls are provided with a layer of
material that prevent measurement of radiation or light directed at
the walls for passing through the side walls. The patent discloses
that a photo-measurement will be taken of each individual cuvette
and the material therein.
The U.S. Pat. No. 4,400,353 to Meseral, etal. discloses an
electro-optical system for use in evaluating immunological
reactions. Fluid biological test specimens and a reagent are
introduced into a reaction zone in an image cell. The reaction
cells are formed of two planar surfaces made of glass or plastic
material which are provided with a generally circular groove to
define the reaction cell. A fill port is pierced in a circular
groove for introducing the reagent and the biological fluid. Each
image cell is lifted out of its respective compartment and brought
into the optical pass sequentially. After transluminating the
reaction zone and imaging light being transmitted therethrough on
an image sensor, the dark areas formed on the surface of the image
sensor are measured by electronics. The image sensor is a charge
coupled device (CCD). When several indicator particles agglutinate,
the resulting image will shadow several pixels which appear darker
than a single particle. The CCD is scanned electronically row by
row to obtain each pixel of information. The image areas are
quantified electronically and the total area is obtained which is a
function of the concentration of the antibody in the wells. The
total dark images of the control specimen is related to the
respective concentrations. The imaged data is fed to a threshold
comparator and particle counter which screens the non-agglutinated
particles on the basis of both intensity and particle size.
The U.S. Pat. No. 4,453,266 to Bacus discloses a method and
apparatus for measuring cell volume of a red blood cell on a slide.
The apparatus includes means for generating signals representative
of the area of the cells, and means for measuring the optical
density of the individual cells and for generating signals
representative of the hemoglobin or massive cells. More than one
red blood cell is determined. The image is obtained by a television
camera which sends this image to electronics for the analysis. Each
of the several cells displayed in the image are independently
analyzed.
The U.S. Pat. No. 4,580,895 to Patel discloses a scanning
photometer for reading agglutination tests and other procedures by
scanning the contents of a micro-test well or other sample holding
vessel to determine certain characteristics of the content. The
patent discloses scanning an entire tray having a plurality of
wells and obtaining a video image across each well. The tray or
plate which is utilized has an array of uniformly diametered,
upwardly opening sample-holding wells. An XY mover is connected to
the holder to move the sensor assembly in a horizontal X-Y
coordinate plane to successively bring wells in a preselected order
to axial alignment with photodetector. The sensor assembly
comprises a photo beam interrupt comb which has a set of parallel
and uniformly spaced apart photo beam interrupting teeth arranged
in a straight row extending parallel to the motion path of the
carriage in X coordinate axis. The assembly also comprises a photo
beam interrupt comb which has eight parallel teeth which extend in
the Y coordinate axis. The combs cause the production of interrupt
signals to the microprocessor to aid in the movement of the tray
and designation of each well. The scanning operation is repeated
column for column of each of the columns in plate. The
photodetector's analog output signal is a measurement of the
intensity of the photometers light beam and represents a
continuous, traveling measurement of the optical density of the
substance in diametrically across each entire well and the optical
density of the well bottom. Twenty-four signal samples of the
photodetector analog output are digitized periodically such that
samples are uniformly spaced apart diametrically across each well.
The digital optical density readings of each of the wells are
processed by the microcomputer. The photodetector obtains a sample
across a diameter of the wells, rather than the entire circle of
the well. The microcomputer utilizes a threshold value in a
determination of lights and darks of the sample.
The U.S. Pat. No. 4,784,947 to Noeller discloses still
photographing a plurality of samples at a single time.
The U.S. Pat. Nos. 4,720,463 and 4,856,073, both to Farber et al.
relate to an apparatus and process of automatically obtaining test
results from microbiological test rays. In general, microbiological
sample and agent to be tested are placed in test rays having a
plurality of wells or cupolas. The trays are moved to an incubator
for a predetermined time. Thereafter, the trays are moved to an
inspection station. A light source is disposed above the tray and a
pair of video cameras are disposed below the tray in the inspection
station. The video cameras take images of the tray, well by well,
and a processor processes the images to analyze the test results.
The processor records the background light level of each point or
pixel only within the area of interest for each particular well of
the tray. For each well, the image processor determines the number
of pixels in the area of interest which have an associated voltage
exceeding a predetermined threshold for that area of interest. If
the number of pixels exceeds a predetermined number, a positive
result is assigned to that well. The image processor analyzes the
binary partial results from the wells to determine possible
identity of the micro-organisms.
The present invention provides a drastic simplification of the
prior art apparatus which more accurately identifies and provides a
susceptibility testing of a sample. The present invention utilizes
a mechanically simple system which utilizes a fluorescent reaction
for our identification and susceptibility testing. The fluorescent
determinations are faster and much more accurate than prior art
determinations due to the high signal to noise ratio of
fluorometric systems. Further, an entire tray including a plurality
of wells can be imaged simultaneously, not requiring a well by well
video inspection. This will increase the speed of inspection, which
will provide adequate time for "real time" detection,
identification and susceptability analysis.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a
diagnostic microbiological testing apparatus for detecting the
presence of a fluorescence emitting reaction (FER) resulting from
the interaction of fluorescence emitting agents (FEA) and a sample
for detection, susceptibility, and identification testing, the
apparatus including a test tray including a plurality of reaction
chambers containing the FEA, which upon reaction with a
predetermined microbe in the sample will emit light at a
predetermined emission wavelength upon being illuminated by light
at a predetermined excitation wavelength. A light source is
disposed proximate to the test tray for directing light, at the
excitation wavelength, at the reaction chamber. Filter means passes
therethrough only light generated by the FER at the emission
wavelength. Video means detects only the light generated by the FER
at the emission wavelength simultaneously from the plurality of
reaction chambers, the filter means being disposed between the test
tray and the video means.
The present invention further provides a method of detecting the
presence of the FER resulting from the interaction of the FEA and a
sample for detection, susceptibility, and identification testing by
containing the FEA in the plurality of reaction chambers, directing
light at the excitation wavelength at the chambers, passing through
a filter only light generated by the FER at the emission
wavelength, and video imaging only the passed through light
generated by the FER at the emission wavelength simultaneously from
the plurality of reaction chambers.
BRIEF DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to
the following detailed description when considered in connection
with the accompanying drawings wherein:
FIG. 1 is an elevational view of an apparatus made in accordance
with the present invention;
FIG. 2 is a top plan view taken substantially along lines 2--2 of
FIG. 1;
FIG. 3 is a plan view of a test tray for use with the present
invention; and
FIG. 4 is an example of susceptability testing used in accordance
with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
A diagnostic microbiological testing apparatus instructed in
accordance with the present invention is generally shown at 10 in
the Figures. The apparatus is specifically useful for detecting the
presence of a fluorescence emitting reaction (FER) resulting from
the interaction of fluorescence emitting agents (FEA) and a sample
for detection, susceptibility and identification testing.
Many FEA have been characterized for use in detection,
susceptibility and identification testing. The U.S. Pat. No.
5,164,301 to Thomson, issued Nov. 17, 1992 and assigned to the
assignee of the present invention discloses a two-dye technology
for detecting, identifying and susceptibility testing of samples.
The technology includes a metabolic dye which changes in response
to such environmental factors as pH or enzymatic cleavage, and an
analytical dye. Examples of such FEA are medias containing
metabolic dyes such as resazurin, indoxyl and chloroindoyl
compounds and analytical dyes such as sulforhodamine, rhodamine B,
eosin V and flourescein. Examples of such media are simple media
chosen to promote the growth of microorganisms tested. The media
may contain carbohydrates. It may be made to grow specific microbes
or a broad spectrum of microbes. Such media are well known in the
art, such as Mueller-Hinton, Columbian broth, Schaedler's broth,
Brain heart broth, and tryptic soy broth.
Samples to be tested in accordance with the present invention and
utilizing the apparatus made in accordance with the present
invention can be various solid or fluid samples taken from a
patient. The samples can be in the form of blood samples, plasma
samples, spinal fluid samples or the like. Of course, the present
invention could be used for veterinary and other purposes.
Generally, the apparatus 10 includes a plurality of reaction
chambers in the form of test tray 11. As shown in FIG. 2, the
apparatus 10 can include a plurality of test trays 11 contained
within a carousel 14 which is effectively a rotary table rotated by
an actuating and indexing mechanism 16. The carousel 14 is rotated
to be able to index one of the test trays 11 proximate to a
detection area 18, as shown in FIG. 2. Positioning means 20, such
as a reciprocating arm mechanism, positions one of the test trays
11, proximate to the detection area 18, into and out of the
detection area for purposes as explained below. That is, the
carousel 14 would allow in and out sliding movement of a test tray
11 as actuated by the positioning mechanism 20 into and out of the
detection area 18.
A light source, schematically shown at 22 in FIG. 2, is disposed
proximate to the test tray 11 disposed in the detection area 18.
The light source 22 is a high energy narrow band light source which
can excite fluorogenic agents at specific bands such as, gas filled
electron discharge tubes or lasers. The light source provides a
high energy narrow band wavelength light sufficient to produce an
emission fluorescence in the presence of a microbe in the sample to
be detectable by the video mechanism 26, as discussed below. A
filter 24 is shown to be disposed on the side of the test tray 11
opposite to the light source 22. The filter 24 is of the type that
passes therethrough only light generated by the FER at the emission
wavelength. That is, the filter 24 filters out light at all other
wavelengths than the emission wavelength from passing therethrough.
Thusly, the only light detectable beyond the filter 24 is light
generated by the FER. All other images, such as an image of the
test tray 11, are not detectable beyond the filter 24. Further, all
of the reaction chambers 12 are detectable if an FER is present
therein. Otherwise, even the reaction chamber 12 is not detectable
as it will emit no detectable light beyond the filter 24.
The apparatus 10 includes a video mechanism 26, the filter 24 being
disposed between the video mechanism 26 and test tray 11. Moreover,
the only light detectable by the video mechanism 26 is the light
which passes through to the filter 24. Thusly, the video mechanism
26 is only exposed to light generated by the FER at the emission
wavelength. By using the filter 24, a video mechanism
(two-dimensional optical detector) such as a diode array or CCD
device can be used which receives, detects, and produces an image
from a broad spectrum of wavelengths but the filter 24 will only
expose the video mechanism 26 to the light emitted by the FER at
the emission wavelength. The narrow band light source provides
sufficient energy so that the light passing through the filter 24
is of sufficient magnitude so as to be detectable by the CCD
device. Thusly, the video mechanism 26 will detect only the light
emitted by the FER and will not image any other objects illuminated
by the light source 22, such as the reaction chamber 12. Further,
the video mechanism 26 will image all FER simultaneously when the
test tray 11 is disposed at the detection area 18. Thusly, a series
of reaction chambers 12 can be imaged, unlike prior art systems
which must scan each reaction chamber separately.
The above components of the apparatus 10 are contained within a
body portion 28, having a lid member 30. The body portion 28 and
lid 30 completely isolate outside light sources from the detecting
system comprising the light source 22, test tray 11, filter 24 and
video mechanism 26.
Since the reactions occurring in the test trays 11 need to be
controlled with regard to temperature, the apparatus 10 can include
a temperature control and display schematically shown at 32 in FIG.
1. The apparatus also includes control electronics schematically
shown at 34 for controlling the operation of the carousel 14,
camera 26 and positioning mechanism 16.
The present invention further provides a method of detecting the
presence of the FER resulting from the interaction of the FEA and
the sample for detection, susceptibility and identification
testing. Specifically, the method includes the steps of containing
the FEA in a plurality of reaction chambers 12, the FEA upon
reaction with a predetermined microbe in a sample contained within
the reaction chamber 12 emitting light at the predetermined
emission wavelength upon being illuminated by light at the
predetermined excitation wavelength. Light is directed at the
excitation wavelength from the light source 22 to the reaction
chambers 12. A filter 24 passes therethrough only light generated
by the FER at the emission wavelength and the passed through light
generated by the FER at the emission wavelength from the plurality
of reaction chambers 12 is simultaneously detected by the video
mechanism 26.
The following example demonstrates the ability of the present
invention to perform susceptibility testing. The inoculum which is
comprised of: growth media (Mueller Hinton base at 22 g/L),
fluorogenic substance (sulforhodamine 101 at 10 .mu.M), reaction
dye (resazurin at 20 .mu.M), antimicrobial agent (Aztreonam at
various concentrations) and Providencia alcalifaciens at
5.times.10.sup.5 cfu/m.iota., is introduced to multiple reaction
chambers with the capacity of 50 .mu..iota. each. The test plate is
incubated by setting the carousel temperature to 35.degree. C., and
amplitude readings are taken every 6 minutes. FIG. 4 illustrates
the time curves of the various samples in which curves #2, 3 and 4
indicate growth of microorganisms due to the low concentration of
antibiotics (neg. control, 4, 8 .mu.g/m.iota..) in the
corresponding chambers. Curve #1 shows the growth inhibition of the
microorganisms in the presence of the higher antibiotic
concentration (the Minimal Inhibition Concentration - MIC) of 16
units.
The invention has been described in an illustrative manner, and it
is to be understood that the terminology which has been used is
intended to be in the nature of words of description rather than of
limitation.
Obviously many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the appended
claims wherein reference numerals are merely for convenience and
are not to be in any way limiting, the invention may be practiced
otherwise than as specifically described.
* * * * *